Project 2: Eph Signaling in Embryonic Motor Neurons Several recent studies have shown that ephrin-A - EphA "forward" and"reverse" signaling are involved in controlling motor neuron axon navigation during embryonic development. This grant is directed at understanding the mechanisms that control how and where ephrin-A - EphA signaling occurs, in order to understand how spinal locomotor circuitry is formed. Cellular and developmental studies of motor neurons have revealed the inductive interactions that trigger their differentiation, the cellular interactions that control their axonal projections, the trophic interactions that support their survival, and the post synaptic interactions that lead to maturation of their synapses. The signaling pathways that actually guide motor neuron axons to their appropriate targets, however, remain poorly defined. During vertebrate development motor neuron subtypes are generated that exhibit distinct cell migration patterns and specific preferences for axon pathways. In this grant we propose to examine how a well defined family of axon guidance molecules, the EphAs-ephrinAs, are used in sophisticated temporal and spatial ways to control the axonal navigation of multiple classes of motor neurons. Genetic studies indicate that EphA4 is used at multiple choice points for motor neuron pathfinding. This appears to be based on a precise spatial localization of EphA4 protein along the proximo-distal axis of specific motor neuron subtypes. In Aim 1 we will investigate whether this localization is mediated by RNAtransport and selective translation, protein degradation, and/or temporal regulation of transcription. In Aim 2 we will examine the mechanisms that control EphA4 expression in motor neurons by investigating a possible connection between the Lim1 (Lhx1) LIM-HD transcription factor and neuronal activity. In Aim 3 we examine how EphA proteins can function as ligands to reverse signal through ephrin-As expressed by motor neurons, focusing on p75NTR as a possible coreceptor. More generally, these studies should provide a better understanding of how a limited number of guidance molecules can be used in diverse ways to wire the CMS.These findings should help to develop innovative methods for restoring motor function lost due to injury or disease.